Induced inactivation of Wnt16 in young adult mice has no impact on osteoarthritis development

Abstract

Osteoarthritis (OA) is a common disorder and a major cause of disability in the elderly population. WNT16 has been suggested to play important roles in joint formation, bone homeostasis and OA development, but the mechanism of action is not clear. Transgenic mice lacking Wnt16 expression (Wnt16-/-) have a more severe experimental OA than control mice. In addition, Wnt16-/- mice have a reduced cortical thickness and develop spontaneous fractures. Herein, we have used Cre-Wnt16flox/flox mice in which Wnt16 can be conditionally ablated at any age through tamoxifen-inducible Cre-mediated recombination. Wnt16 deletion was induced in 7-week-old mice to study if the Cre-Wnt16flox/flox mice have a more severe OA phenotype after destabilizing the medial meniscus (DMM surgery) than littermate controls with normal Wnt16 expression (Wnt16flox/flox). WNT16 deletion was confirmed in articular cartilage and cortical bone in Cre-Wnt16flox/flox mice, shown by immunohistochemistry and reduced cortical bone area compared to Wnt16flox/flox mice. After DMM surgery, there was no difference in OA severity in the articular cartilage in the knee joint between the Cre-Wnt16flox/flox and Wnt16flox/flox mice in neither female nor male mice. In addition, there was no difference in osteophyte size in the DMM-operated tibia between the genotypes. In conclusion, inactivation of Wnt16 in adult mice do not result in a more severe OA phenotype after DMM surgery. Thus, presence of WNT16 in adult mice does not have an impact on experimental OA development. Taken together, our results from Cre-Wnt16flox/flox mice and previous results from Wnt16-/- mice suggest that WNT16 is crucial during synovial joint establishment leading to limited joint degradation also later in life, after onset of OA. This may be important when developing new therapeutics for OA treatment.

Fig 1. Immunohistochemistry and μCT reconstructions.

(A) Representative images of immunohistochemical staining of WNT16 in the articular cartilage in tibia, showing WNT16 protein expression in Wnt16flox/flox but not Cre-Wnt16flox/flox mice. (B) The tibial subchondral bone of the Wnt16flox/flox and Cre-Wnt16flox/flox control knees (Control) and the knees that underwent DMM surgery (DMM). The arrows point at osteophytes.

Fig 2. Subchondral bone.

Two-way ANOVA was used to assess the effects of surgery (control vs. surgery destabilizing the medial meniscus [DMM]) and genotype (Wnt16^flox/flox vs. Cre-Wnt16^flox/flox) on bone parameters. There was no effect on the total medial subchondral bone volume/tissue volume (BV/TV) by DMM surgery or genotype in female (A) or male (B) mice. In both females (C) and males (D), DMM surgery decreased the trabecular thickness (Tb.Th) but the genotype had no effect. The Trabecular number (Tb.N) was increased by DMM surgery in both female (E) and male (F) mice, but the genotype had no effect. P < 0.05 was considered statistically significant. (Females N = 10–12; males N = 11).

Fig 3. OARSI scores for control knees and knees that underwent DMM surgery in female mice.

(A) The summed OARSI scores for the total knee (total knee = medial tibial plateau [MTP] + medial femoral condyle [MFC] + lateral tibial plateau [LTP] + lateral femoral condyle [LFC]), where the maximum score for the total knee is 24. (B) The OARSI score for the MTP and the MFC. The maximum score for each region is 6. (C) The OARSI score in the LTP and LFC. The maximum score for each region is 6. (D) Representative microphotographs of the MTP and MFC in control and DMM-operated knees in Wnt16^flox/flox and Cre-Wnt16^flox/flox mice. Especially the MTP in the DMM-operated knees show cartilage damage in both the Wnt16^flox/flox and Cre-Wnt16^flox/flox female mice. Magnification × 20 and the black bar in the photos represents 100 μm. Data are shown as scatter plots where the bars show the mean. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, the Mann-Whitney U test was used (N = 10/group for the Wnt16^flox/flox and N = 12/group for the Cre-Wnt16^flox/flox mice).

Fig 4. OARSI scores for control knees and knees that underwent DMM surgery in male mice.

(A) The summed OARSI scores for the total knee (total knee = medial tibial plateau [MTP] + medial femoral condyle [MFC] + lateral tibial plateau [LTP] + lateral femoral condyle [LFC]), where the maximum score for the total knee is 24. (B) The OARSI score for the MTP and the MFC. The maximum score for each region is 6. (C) The OARSI score in the LTP and LFC. The maximum score for each region is 6. (D) Representative microphotographs of the MTP and MFC in control and DMM-operated knees in Wnt16^flox/flox and Cre-Wnt16^flox/flox mice. Especially the MTP in the DMM-operated knees show cartilage damage in both the Wnt16^flox/flox and Cre-Wnt16^flox/flox female mice. Magnification × 20 and the black bar in the photos represents 100 μm. Data are shown as scatter plots where the bars show the mean. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, the Mann-Whitney U test was used (N = 10/group for the LFC and total lateral score in the control Wnt16^flox/flox mice, N = 11/group for the medial scores in the control Wnt16^flox/flox mice, N = 11/group for the DMM-operated Wnt16^flox/flox mice, and the Cre-Wnt16^flox/flox mice).